An investigation
was performed on the possibilities for an alteration of the total solidification
temperature when preserving concentrated salt media (MBM, MEM, 199, HAM), used
for the maintenance of animal viruses and cell cultures. It had been established
that by the use of dextran 40000, this temperature could be increased with about
20°C. A protection in 3.6% concentration was recommended and the freezing
to minus 40°C might be performed directly in the refrigerator for storage.
The temperature should not be raised over minus 32°C during the stockage.

Introduction

The maintenance of animal viruses and cell cultures is related
to their multiplication in biological hosts and cryogenic conservation. It is
accompanied by the application of synthetic media based on balanced salt solutions.
These solutions, according to the investigations of G. Benet and J. M. Preaud
[2], require special attention for the preservation of their qualities and homogeneity.
The quantities of the media used in the collecting activity are comparatively
small, both in the solutions (series) prepared in the labs and as industrial
packing. On the other hand, the application of a controlled series nutrient
medium is recommended for the achievement of standardisation in the work as
long as possible. The decision can be found in the preparation of concentrated
media that divided into portions, to be stored as frozen. We have been using
for years this way to standardise the conditions for cell cultures and viruses
maintenance. It has to be mentioned that the total solidification temperature
of the basic salt solutions is low. The ten-fold concentrated media completely
freeze up at temperatures lower than minus 50°C - Table
1 [2]. This necessitates the application of low-temperature freezers providing
minus 70°C for the conservation.

On the other side, the eutectic temperature
of the quoted media is high and enables their storage in "low class" refrigerators
providing temperature of minus 40°C.

The low total solidification temperature
for virus vaccines, obtained with these media, also brought problems to us during
their lyophilization. The freezing time was prolonged, thence - the time for
the vacuum sublimation drying. The production appeared to be more expensive.
We realised, that by the cryoprotection, aiming at maximal preservation of the
infectivity when lyophilizing viruses, the dextrans increased the full solidification
temperature of the suspensions [3]. This gave us a possibility to work out and
introduce a new lyophilization technology for many vaccines [4].

Meeting again the problem for the
cryopreservation of salt solutions and nutrient media for the purposes of virology,
we set us the task of making detailed investigations on the possibility to increase
their total solidification temperatures.

Materials and Methods

We provided the same
apparatus, methodology and technical performance for to achieve comparability
in the accomplished results with the ones of the scientists who had ever described
such an investigation - G. Benet and J. M. Preaud [2].

Media. Five of the
media were prepared and filtered through a membrane filter 0.22 mm:

Excel was the program used
for building up the graphic dependencies and deducing the results.

Working methods. The
method of O. Rey [9] had been used. It was based on the resistance changes,
depending on the temperature of the studied medium when freezing and melting.
Six millilitres of the material were placed in a special glass chamber, provided
with drills for estimating the resistance and the temperature. The sample was
frozen down to minus 70°C at a speed of 2°C.min-1 in an
ultracryostat "Nikol". A styropor chamber with a wall thickness of 5 cm, providing
a smooth increase of the temperature at a speed of 1 to 2°C.min-1,
was used for the melting. The results achieved by five independent measurings
were processed on a PC and graphics were build up. The temperature, at which
a sharp rise in the resistance when freezing the sample had been registered
- 15 to 20 MW.cm, was considered to be the total
solidification one. The temperature at a resistance value of 0.01 MW.cm,
achieved during the melting, was considered to be the eutectics[2].

We included observations about the
appearance of side effects on the development of over 20 different cell cultures,
cell lines and viruses, caused by dextran, as an additional investigation.

Results and Discussion

The addition of dextran
to the different media obviously increased their total solidification temperature.
The initial experiments made clear that the effective dextran concentration
as a cryoprotector was 3% to 4%. Higher concentrations (up to 10%) insignificantly
increased the total solidification temperature and their application was unreasonable
from an economic point of view. On the other side, the physiological concentration
of the dextran when used as a substitute in the blood plasma, had been 3.6%
[1]. On the basis of these data, we continued our investigations with the isotonic
concentration of the dextran - 3.6%.

The generalised results from the performed studies for the total solidification
temperature of the investigated media determination, after the addition of dextran,
are presented in Table2. When comparing
the values with the ones in Table 1, we
established that the total solidification temperature increased with more than
20°C for all the samples.

No change was registered in respect
to the eutectic temperatures.

The results from the biological experiments
exhibited a complete indifference of the dextran in the nutrient media, towards
the multiplied cell cultures and viruses (data not shown).

Conclusions

The accomplished investigations
and the achieved results give us a reason to recommend a protection to be performed
with dextran (40000) in a concentration of 3.6%, when preserving concentrated
media like BME, MEM, 199, HAM as frozen.

The cooling speed does not influence
the process, so the freezing to minus 40°C may be performed directly in
the refrigerator for storage.

The temperature is recommended not
to be raised over minus 32°C during the stockage.